The aim of this project is to elucidate the mechanism for entry of the anti-cancer drug cis-diamminedichloroplatinum II (cisplatin) into cancer cells and to determine how drug resistance affects the subcellular distribution of cisplatin. Drug-sensitive and drug-resistant liver and epidermoid carcinoma cells were incubated with cisplatin at concentrations up to 400 micromolar for periods up to 4 hours. The enzyme trypsin was used to release the cells gently from the culture dishes. Serum was subsequently added to stop the trypsin activity and to allow the cells to recover their sodium/potassium gradient. Cells were then pelleted, rapidly frozen in liquid ethane and cryosections cut to a thickness of 100 nm. Sections were picked up on copper grids and stored under liquid nitrogen prior to analysis. Specimens were cryo-transferred into a FEI CM120 transmission electron microscope or a VG Microscopes HB501 STEM equipped with energy-dispersive x-ray spectrometers for electron probe x-ray microanalysis. In the absence of cisplatin, cells prepared in this way were found to have normal Na/K gradients across the plasma membrane. Experiments are currently being performed on treated cells to localize cisplatin within intracellular compartments. Sections are also being sent to Argonne National Laboratory for analysis by synchrotron-based x-ray microprobe. (This project is a continuation of Intramural Research Project 1 Z01 OD 010491-03.)